ANTIGEN PROCESSING AND PRESENTATION

CHAPTER 9

ANTIGEN PROCESSING AND PRESENTATION B.M. Chain, L Sealy, D.R. Katz and M. Binks

The study of antigen processing and presentation, by which we mean the complete ensemble of events leading up to the activation of an antigen-specific T lymphocyte, has been a focus of analysis by immunologists since the discovery of the T cell itself, and indeed even before. Three characteristics have maintained the study of these particular interactions in the forefront of research over more than 30 years. First, the increasing realization that the antigen-presenting event is a key point in the regulation of the immune system; secondly, the high degree of complexity of the antigen-presenting event; and thirdly, the continuing interdisciplinary nature of antigen-presentation research. Thus the field was dominated initially by classical genetics, then by the development of sophisticated in vitro cell culture techniques, by the rapid expansion of molecular genetics, and, most recently, by newer technological advances in cell biology, crystallography and peptide analysis. An overview illustrating the tremendous breadth of the field of antigen processing and presentation is shown in Table i, together with a few of the more recent reviews covering each topic. T a b l e 1. Specialties and subspecialties in antigen processing and presentation Refs Genetics M H C locus M H C and disease

1 2, 3

Intracellular biology Antigen uptake Antigen degradation Intracellular cycling MHC/peptide interaction

4 5, 6 6-8 9-11

Intercellular biology Antigen-presenting cell heterogeneity Intercellular interaction

12-14 15-17

Integrating the system Antigen presentation and the repertoire

18

Therapeutic intervention MHC/peptide/TCR interaction Accessory molecules

19 20, 21

ANTIGEN PROCESSING AND PRESENTATION

173

The aim of the present compilation is neither to review the subject, nor to provide an exhaustive bibliography. Still less is it possible to collect together all the information now available about antigen-processing systems. Rather, we have sought to draw together collections of information regarding some of the best-studied aspects of antigen processing/ presentation, which may be of value to those trying to establish experimental systems of their own, or those seeking illustrative examples of our knowledge of this field.

1. GENETICS Genetics, both classical and molecular, has played a key role in understanding antigen processing and presentation. The most recent impetus in this area has come from the application of transgenic technology, and dozens of novel transgenic strains, either overexpressing antigens or intrinsic molecules of the immune system, or using homologous recombination to 'knock-out' specific gene expression, are now available. The essential observation that T-cell recognition can be quantitatively and qualitatively altered by a multitude of host genes still seeks final explanation. Nevertheless, attention for more than three decades has focused on the genes of the major histocompatibility complex (MHC, known as human leukocyte antigen (HLA) in humans, or the H-2 region in mouse), which regulate the magnitude and nature of all T-cell-dependent responses. This region of the chromosome has been studied intensively in both animals and man, the most recent impetus coming from the powerful techniques of chromosome walking. This analysis has revealed that the MHC, as a locus, contains many genes with as yet no known function and others with no obvious role in antigen processing and presentation. The MHC is covered in Chapter 10, and therefore a genetic map of the human MHC showing only those genes believed to have an antigen processing/presentation function is shown in Figure 1. The fine details of the maps continue to change, and novel genes within the MHC region still remain to be discovered, but the general outline of the map is unlikely to alter substantially. A unique feature of the MHC locus is its high degree of polymorphism. This makes analysis of immune responses in human populations very difficult. Hundreds of alleles have already been described, and more are continually reported as analysis moves from serology to sequence; the sequences of many human and mice alleles are already available in sequence databanks such as that provided by the EMBL database. For this reason, the study of antigen processing and presentation has been helped enormously by the availability of inbred mouse strains homozygous at the MHC. Many of the commonly used strains, as well as a number of MHC recombinants , are illustrated in Table 2. Of particular value are the sets of congenic strains, which carry different sets of MHC alleles on identical genetic backgrounds. Comparison of immune responses in such mice allows the identification of the MHC contribution to immune response regulation to be determined directly. Despite the importance of the MHC, it is important to note that in the case of most responses to complex antigens, regulation by non-MHC polymorphic loci, many of which remain to be identified, are of considerable importance.

2. THE ANTIGENS A second approach to the study of antigen processing and presentation has been the detailed dissection of T-cell epitopes within well-characterized model protein antigens. A key element of these studies has been the use of short synthetic peptides to identify the site recognized by in vitro cultured clonal populations of antigen-specific T cells. Indeed, the demonstration that such short linear epitopes can substitute for the intact protein antigen (in sharp contrast to the predominantly configuration-dependent epitopes recognized by antibody) was itself a major

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Figure 1. Map of the human MHC, showing the genes involved in antigen processing and presentation (adapted from ref. 1). DPB1 DPA2 DPA1 i I / DPB2\ I //

TAPI LMP7 DQB2 APd TAP2 \\ I| 7 I DQA2 DMADMB \ \ I /DOB I/DQB3 L MP2 LMP2

DNA

D 000

D

00 1 1 1 ! 001

1^ 400

I 300

600

500

DQB1 I DQA1

00

DD D

10

T" 900

800

700

DRA ™ DRB9 I

DRB2 DRB1 I DRB3

1000

CLASS II Hsp70 17 B

2 IHom

1100

~i

r

1600

1700

Φ

II 2100

2000

2200

-fh

2600

2700

CLASS III

30

92

I I

2800

3100

3000

2900

I 3300

3200

80

I

I

3400

CLASS I

21 A 170

G 90 75

16 H

II

III

3500

I

III 3600

H

3700

class I genes

[HI ABC transporter genes 3800

^

proteosome-like genes

Ξ

Hsp70

Ö

class II a and β genes

landmark in identifying the nature of the antigen-processing event. Several dozen antigens have been analyzed in this way. An analysis of some of the best-characterized T-cell epitopes identified by T lymphocytes within a very small set of intensively studied proteins is shown in Table 3. In the case of class II MHC restricted epitopes, the epitope can be mimicked by peptides of a variety of different lengths, and the region shown is only an approximation to the 'natural' peptide(s) produced by the antigen-presenting cell. In the case of class I peptides, the length restrictions are much tighter, and a very sharp fall in potency is often observed when using longer than optimal peptides. In both cases, presentation of specific epitopes is largely, although not absolutely, restricted to particular alleles of specific MHC molecules. A second key feature is that the range of epitopes actually recognized by T cells immunized to

ANTIGEN PROCESSING AND PRESENTATION

175

T a b l e 2. I n b r e d m o u s e strains commonly used in immunological studies (data taken from ref. 22) The mouse strains representing the major M H C haplotypes in inbred laboratory strains 1 H-2 b C57B1/10, C57B1/6 H-2 d DBA/2, BALB/c H-2 k C3H, CBA, AKR H-2 P B10.P H-2 q DBA/1, B10.Q, B10.G H-2 r B10.RIII, RIII H-2 S A.SW, B10.S H-2 Z NZW Congenic sets of M H C Mouse strains BALB/c BALB.B10 BALB/AKR BALB.K

matched mouse strains 2 H-2 haplotype d b k k

Mouse strains C3H C3H.B10 C3H.NB C3H.SW

H-2 haplotype k b P s

AKR AKR.B6/1 or 2

k b

C57B1/6 C57Bl/6bm set

b b mutants 3

C57B1/10 C57B1/10.AKR C57B1/10.ASW C57Bl/10.Br C57B1/10.NB C57B1/10.Q C57B1/10.NZW C57B1/10.RIII

b k s k P q z r

C57B1/10.CBA C57B1/10.CNB C57B1/10.D1 C57B1/10.D2 C57B1/10.F C57B1/10.G C57B1/10.S C57B1/10.M

k P q d P q s f

^ach designated strain represents an independently derived line, expressing homozygous MHC genes of the appropriate haplotype at all MHC class I and II loci. 2 Each set of strains expresses the appropriate different sets of MHC genes in the context of an identical background provided by the designated parental strain. Thus all C57B1/10 congenics express the same complex of non-MHC genes, and differ only at the MHC locus. Each strain has been obtained by crossing two homozygous MHC disparate strains and then repeatedly backcrossing against the background parental strain. 3 This set of mouse strains expresses the same MHC genes, but with a variety of well-characterized small mutations in the parental H-2b genes. an intact protein antigen is usually very small, and that many factors, including the affinity of processed peptides for M H C , selection by the processing machinery itself (e.g. the specificity of enzyme cleavage sites) and interantigenic competition, all play a part in shaping the population of responding T cells. More recently, a complementary approach to the identification of T-cell epitopes has become possible, involving the elution and sequencing of MHC-bound peptides. A major advantage of this system is clearly the ability to identify directly the 'naturally' processed epitope. However, the technology required for the micro-isolation and sequencing is still very complex and costly, and this has limited the number of laboratories able to carry out such analyses. The

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CELLULAR IMMUNOLOGY LABFAX

Table 3. Some T-cell epitopes identified by peptide analysis of T-cell responses Antigen

Sequences

Restriction element

35-43 46-61 116-129 74-86 94-100 20-28 38-45 52-69 81-96 1-18 25-43 107-116 31-50 93-113

I-A k I-A k I-A k I-A k I-A k I-A b I-A b I-A b I-A b I-E k I-E k I-E d I-E q I-E q

Ovalbumin

323-339 257-264

I-A d Kb

Myoglobin

108-117 132-146 112-118 37-53 61-77 73-101 109-125 133-149

I-A d I-E d I-E d SJL class SJL class SJL class SJL class SJL class

Lysozyme

Tetanus toxoid

830-843 953-967 947-960 949-960 1273-1284

Comments

Dominant Cryptic Cryptic

Dominant

II II II II II

DR5 DR5 DR7 and DR9 DP2 and DP4 DR52a/52c

Promiscuous

Influenza hemagglutinin HA1 Strain H3N2/AX31

56-76 71-91 81-97 177-199 186-205 206-227 257-271 54-62 68-83 120-139 226-245 246-265

I-A d I-A d I-A d I-A d I-A d I-A d I-A d I-A k I-A k I-A k I-E k I-E k

ANTIGEN PROCESSING AND PRESENTATION

177

T a b l e 3. C o n t i n u e d Antigen

Sequences

Restriction element

A/JAP/57

202-212 211-221

H-2 d class I H-2 d class I

A/PR/8/34

523-545 259-266

Kk Kk

Comments

For further details see refs 23-26. Dominant epitope: the major epitope recognized in the particular antigen/strain combination. Cryptic epitope: an epitope recognized in isolation, but not recognized when presented to the immune system in the context of the whole antigen molecule. Promiscuous epitope: an epitope recognized in the context of several different HLA types. published data on peptides identified so far, which identify only a tiny percentage of the peptides actually bound to any individual M H C haplotype at one time, are listed in Tables 4-7. In parallel with this development has been the ability to use sequencing to identify 'conserved' residues, or motifs, which can be used to predict likely T-cell epitopes within a complex protein. It is important to realize, however, that these predictive motifs should only be used as a guide. There are examples of peptides with motifs not being recognized, since M H C peptide binding is necessary but not sufficient for a response. Conversely, peptides with low affinity for M H C (whose M H C binding cannot be readily detected) may function as strong T-cell epitopes. Prediction cannot, at present, replace the more laborious task of identifying epitopes experimentally.

3. ANTIGEN-PRESENTING CELLS The stimulation of C D 4 + T cells, which are the predominant regulatory element of the immune system, occurs during interaction with a class II bearing antigen-presenting cell. Class II M H C expression under normal conditions is restricted to rather few cell types, including the lymphoid (interdigitating) dendritic cell and its related family, the B cell, and the thymic epithelium. This latter is believed to play an important role in thymic eduction, probably by regulating 'positive selection'; rather little is known about the physiology or cell biology of this process. Certain macrophage subpopulations appear to express low levels of class II M H C constitutively, and the expression of class II on macrophages is readily upregulated by a number of inflammatory mediators. Other cell types, including cells of epithelial and endothelial origin, have been shown to express class II M H C molecules either in vivo, during strong T-cell-dependent inflammatory responses, or in vitro under the influence of cytokine regulation. The physiological function of this 'aberrant' class II expression is still debated, and this class of cells are collectively termed 'nonprofessional' antigen-presenting cells. The characteristics of the three major antigen-presenting cell types are described below.

3.1. The interdigitating (lymphoid) dendritic cell

The lymphoid dendritic cell forms part of an antigen-presenting cell family, comprising the Langerhans cell of the skin, the veiled cell of the lymph, and the interdigitating cell of lymphoid tissue. In addition, related cells are present within surface epithelia and most solid organs. Together this bone marrow-derived family comprises the 'professional' antigenpresenting cell population of the body.

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CELLULAR IMMUNOLOGY LABFAX

Table 4. Sequences of peptides eluted from MHC molecules: human class I Allele HLAA2.1

HLAA2.5

HLAB27

a

Peptide sequence 1 2 3 4 5 6 7 8 9a Anchor residues Frequent residues Ligands

L

Anchor residues Ligands

K G P P F A R P A D

GX G V T A A V TAAVQA X V S V I V E L A X E V V X X X I V H I P Y E V

Human pp30 signal peptide Human pp30 signal peptide p61 (regulatory subunit of PP2A) 9

L V L I Q

Y G V I P E Y F D L I I

K

R R R R R R G f K R R A

R R R R R R R R R R R

Ref. 9

V

ME V K S X P S L L D V LLL D V G X V P S L L P S X X V K X N E Y L L P τ L WV

Anchor residues Frequent residues

Protein source

9 Y Q I K V K WL S K I D Y N F E F T I S L F

K E E P E K G G R G G

s

I V A I P L L P V I

T V V G T I i T E D R

E K K d V L H Q H R A

L K k a R K r R Y K

Histone H3 H3.3 Hsp 89a Hsp 89ß Elongation factor 2 RNA helicase Ribosomal protein Rat 60S ribosomal protein -

Lower-case letters indicate residues of lower confidence.

Antigen-presenting-cell function All members of the family are strong stimulators of CD4 and CD8 T-cell responses, including alloresponses, responses to soluble and particulate foreign antigens, and hap tens. These cells are the only ones capable of initiating a primary T-cell immune response. Dendritic cells in the thymus are believed to regulate 'negative' selection, in the acquisition of self-tolerance. Langerhans cells, but not dendritic cells of lymphoid tissue, are phagocytic and express receptors for the Fc region of Ig. However, dendritic cells may be capable of processing particulate antigens by cell-membrane proteinases.

ANTIGEN PROCESSING AND PRESENTATION

179

T a b l e 5. Sequences of peptides eluted from M H C molecules: h u m a n class II Allele

Protein source

Peptide sequence

Ref.

HLA-DR1

Consensus HLA-A2 (105-117) Invariant chain (105-118) ( N a + K + ) ATPase (199-216) Transferrin receptor (680-696) Bovine fetuin (56-73)

1XXXX2XXX3 3 SDWRFLRGYHQYA KMRMATPLLMQALP

27

HLA-DR2

HLA-DQoc chain (97-119) HLA-DQP chain (42-59) HLA-DR2bß chain (94-111) FnRot chain (586-616) K + -channel protein (173-190) Mannose binding protein (174-193) M E T (59-81) GPB-2 (434-450) Apo B-100 (1200-1220) Factor VIII (1775-1790)

HLA-DR3

180

HLA-A30 (28-?) HLA-DRa chain (111-129) Invariant chain (131-149) Acetylcholine receptor (289-304) Glucose transporter (459-474) N a + channel protein (384-397) CD45 (1071-1084) I F N receptor (128-148) EBV gp220 (592-606) IP-30 (38-59) Cyt-65 (155-172) Apo B-100 (1273-1295) (1207-1224) (1794-1810)

IPADLRIISANGCKVDNS RVEYHFLSPYVSPKESP Y K H T L N Q I D S VK VWPRRP NIVIKRSNSTAATNEV(PEVTVFS)

28

(S)DVGVYRAVTPQGRPD(AE) RVQPKVTVYPSKTQP(LQH) LSPIHIALNFSLDPQAPVDSHGLR PALHYQ DGILYYYQSGGRLRRPV(N) IQNLIKEEAFLGITDEKTEG EHHIFLGATNYIYVLNEEDLQKV QELKNKYYQVPRKGIQA FPKSLHTYANILLDRRVPQ(TD) LWDYGMSSSPHVLRNR Q D D T Q F V R F D SD AASQ.. . b PPEVTVLTNSPVELREPN(V)

28

ATKYGNMTEDHVMHLLQNA VFLLLLADKVPETSLS TFDEIASGFRQGGASQ YGYTSYDTFSWAFL GQVKKNNHQEDKIE GPPKLDIRKEEKQIMIDIFH(P) TGHGARTSTEPTTDY SPQALDFFGNGPPVNYKTG(NL) GFAIRPDKKSNPIIRTV (IPD)NLFLKSDGRIKYTL(NKNSLK) YANILLDRRVPQTDMTF VTTLNSDLKYNALDLTN

CELLULAR IMMUNOLOGY LABFAX

T a b l e 5. C o n t i n u e d Allele

Protein source

Peptide sequence

Ref.

HLA-DR4

HLA-A2 (28-50) HLA-Cw9 (28-50)

( VDD)TQFVRFD SD AAS(QRMEPRAP) ( VDD)TQFVRFD SD AASPR (GEPRAPWV) (D)LRSWTAADTAAQIT(QRKWEAA) DLSSWTAADTAAQIT(QRKWEAA) GSLFVYNITTNKYKAF(LDKQ) SPEDFVYQFKGMCYF

28

(130-150) HLA-Bw62 (129-150) VLA-4 (229-248) HLA-DQ3.2ß chain (24-38) PAI-1 (261-281) Cathepsin C (151-167) IgG heavy chain (121-?) Bovine hemoglobin (26-41) HLA-DR7

HLA-A29 (234-261) HLA-B44 (83-99) HLA-DRa chain (101-126) (58-78) HLA-DQoc chain (179-?) 4F2 (318-338) L I F receptor (854-866) Thromboxane-A synthetase (406-420) K + -channel protein (492-516) Hsp70 (38-54) EBV MCP (1264-1282) Apo B-100 (1586-1608) (1942-1954) (2077-2089) Complement C9 (465-483)

HLA-DR8

HLA-DRa chain (158-180) HLA-DPß chain (80-92) LAM Blast-1 (88-108) (129-146)

AAPYEKEPVLSALTNILS(AQL) YDHNFVKAINADQKSW(T) GVYFYLQWGRSTLVSVS.. . b AEALERMFLSFPTTKT (RPAGD)GTFQKWASVVV (PSGQEQRYTCHV) RETQI SKTNTQTYRE(NL)

28

RSNYTPITNPPEVTVLTNSPVELREP GALANIAVDKANLEIMTKRSN SLQSPIVTEWRAQSESAQSKWLS GIGGFVL... b VTQYLNATGNRWCSWSL(SQAR) TSILCYRKREWIK PAFRFTREAAQDCEV GDMYPKTWSGMLVGALCALAGVLTI TPSYVAFTDTERLIG(DA) VPGLYSPCRAFFNK(EELL) KVDLTFSKQHALLCS(DYQADYES) FSHDYRGSTSHRL LPKYFEKKRNTII APVLISQKLSPIYNLVPVK SETVFLPREDHLFRKFHYLPFLP

28

RHNYELDEAVTLQ (DPS)GALYISKVQKEDNSTYI DPVPKPVIKIEKIED(MDD)

ANTIGEN PROCESSING AND PRESENTATION

181

T a b l e 5. Continued Allele

Protein source

Peptide sequence

Ig κ chain (63-80) LAR (1302-1316) L I F receptor (709-726) IFN-oc receptor (271-287) IL-8 receptor (169-188) Ca 2 + release channel (2614-2623) CD35 (359-380) CD75 (106-122) Calcitonin receptor (38-53) TIMP-1 (101-118) TIMP-2 (187-214)

FTFTISRLEPEDFAV(YYC) DPVEMRRLNYQTPG YQLLRSMIGYIEELAPIV

PAI-1 (378-396) (133-148) Cathepsin E (89-112) Cathepsin S (189-205) Cystatin SN (41-58) Tubulin oc-1 chain (207-223) Myosin β heavy chain (1027-1047) oc-enolase (23-?) c-myc (371-385) K-ras (164-180) Apo B-100 (1724-1743) (1780-1799) (2646-2664) (2885-2900) (2072-2088) (4022-4036) Bovine transferrin (261-281) von Willebrand factor (617-636)

Ref.

GNHLYKWKQIPDCENVK LPFFLFRQAYHPNNSSPVCY RPSMLQHLLR DDFMGQLLNGRVLFPVNLQLGA IPRLQKIWKNYLSMNKY EPFLYILGKSRVLEAQ (NR)SEEFLIAGKLQDGLL(H) QAKFFACIKRSDGSCAWYR (GAAPPKQEF) DRPFLFVVRHNPTGTVLFM MPHFFRLFRSTVKQVD QNFTVIFDTGSSNLWV(PSVYCTSP) TAFQYIIDNKGIDSDAS DEYYRRLLRVLRAREQIV EAIYDICRRNLDI(ERPT) HELEKIKKQVEQEKCEIQAAL AEVYHDVAASEFF... b KRSFFALRDQIPDL RQYRLKKISKEEKTPGC KNIFHFKVNQEGLKLS(NDMM) YKQTVSLDIQPYSLVTTLNS (S)TPEFTILNTLHIRSFT(ID) SNTKYFHKLNIPQLDF LPFFKFLPKYFEKKR(NT) WNFYYSPQSSPDKKL DVIWELLNHAQEH(FGKDKSKE) IALLLMASQEPQRM(SRNFVR)

a The putative HLA-DRl peptide binding motif includes three key amino acids; 1, positively charged; 2, hydrogen-bond donor; 3, hydrophobic residue. b Partial sequence not verified by mass spectrometry. LAM = L-selectin (CD62L); LCMV, lympho-choriomeningitis virus; LIF, leukemia inhibitory factor; PAI, plasminogen activator inhibitor; TIMP, tissue inhibitor of metalloproteinase; VSV, vesicular stomatitis virus. Brackets indicate the presence of a nested set of peptides in addition to the basic core sequence.

Table 6. Sequences of peptides eluted from M H C molecules: mouse class I Allele H-2K d

Peptide sequence 1 2 3 4 5 6 7 8 9 Anchor residues Frequent residues Ligands

Y

N P MK T I F N L T Y Q R T R A L V

Anchor residues Frequent residues

Ligands H-2K b

N M MI K L I LE F P Q V V A S N E N ME T M

Anchor residues Frequent residues Ligands

F Y

H I Y E F P Q L H-2K k

H

_2Kkmi

H-2L d

Anchor residues Frequent residues

Anchor residues Frequent residues Ligands

E

K N Y M

E K

Influenza nucleoprotein (147-155) Protein kinase JAK1 Tumor antigen of P815 Listeriolysin {Listeria monocytogenes) (91-99) 9

Influenza nucleoprotein (366-374) 9

L

Y M R G Y V Y Q G L S I I N F E K L

Ref. 9

L I

S Y F P E I T H I K Y Q A V T T T L G Y K D G N E Y I

H-2D b

Protein source

VSV (52-59) Chicken ovalbumin (257-264) Self protein of P815

I

9

I

9

P Q A S G V Y MG Y P H F MP T N L I S T Q N H R A L L S P F P F D L

LCMV nucleoprotein (119-126) pp89 (168-176) tum-antigen P91A (190-198) Mouse spleen protein

9

JAK, Janus kinase; LCMV, lympho-choriomeningitis virus.

ANTIGEN PROCESSING AND PRESENTATION

183

T a b l e 7. Sequences of p e p tides eluted from M H C molecules: m o u s e class II Allele

Protein source

Peptide sequence

Ref.

I-A s

Consensus MuLV env protein IgG2a IgG2a TfR

XXXXITXXXXHXXX IRLKITDSGPRVPIGpn WPSQSITCNVAHPASST NVEVHTAQTQTHREDY KPTEVSGKLVHANFGT XPYMFADKVVHLPGSQ

29

I-A b

Consensus MuLV env I-E a chain Invariant chain I-Aß chain IgG VH

XXNX XXXXPXXXX HNEGFYVCPGPHRP ASFEAQG ALANIAVDKA KPVSQM R M A T P L L M R RPDA EYWNSQPE XNA D F K T P A T L T V D k p NYNA YNATPATLAVD

29

I-E

Consensus MuLV env Bovine serum albumin

29

DR consensus

XXYLYXXXXRRXXYX PSYVYHQFERRAKYK GKYLYEIARRHPYFyap QSYLIHEXXXIS AAYAAAAAAKAAA

I-A d

Apo E Cys C I-E d a chain ApoE Invariant chain Transferrin receptor Ovalbumin λ repressor

WANLMEKIQASVATNPI DAYHSRAIQVVRARKQ ASFEAQGALANIAVDKA EEQTQQIRLQAEIFQAR KPVSQMRMATPLLMRPM VPQLNQMVRTAAEVAGQX ISQAVHAAHAEINE LEDARRLKAIYEKKK

30

I-A k

Hen egg lysozyme Hsp70 I-A k β chain s30 ribosomal protein s30 ribosomal protein Ryudocan

DGSTDYGILQINSRWW IIANDQGNRTTPSY TPRRGEVYTCHVEHP KVHGSLARAGKVRGQTPKVAKQ AGKVRGQTPKVAKQEKKKKKT EPLVPLDNHIPENAQPG

31

I-E d

Hen egg lysozyme

AWVAWRNRCK

32

MuLV, murine leukemia virus. Cell-surface markers Lymphoid dendritic cells express high levels of both class I and class II antigens, and in addition constitutively express high levels of a variety of co-stimulatory and adhesion molecules, including B7, CD54, CDlla/18 and CD58. They do not synthesize I L - 1 , or indeed any other known cytokine. They express low levels of CD4 and hence are susceptible to infection by HIV-1.

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Differentiation The cells of the lymphoid dendritic cell family are believed to be related in a differentiation pathway: Langerhans cells migrate out of the skin, and migrate through the lymphatics (in the form of veiled cells) to lymphoid tissue, where they differentiate into interdigitating dendritic cells. The spleen and lymph nodes may also contain a second resident population which functions to trap and process antigens passing through the tissue. The signals which regulate this differentiation pathway are still not completely elucidated, but the cytokines GM-CSF, TNFoe, IL-1 and IL-4 have all been shown to modulate dendritic cell maturation in vitro. Methods of isolation A wide variety of isolation methods have been reported in the literature, but all exploit the relatively low density (high cytoplasmic to nuclear ratio) of dendritic cells during density fractionation, and negative selection using antibodies to B, T and macrophage markers. Detailed methods can be found in refs 12 and 33.

3.2. The macrophage The macrophage/monocyte family comprises a group of bone marrow-derived phagocytic cells, which are found populating all major tissues and body cavities, and whose major role is scavenging and degrading senescent or damaged 'self, any foreign particles which penetrate the internal environment of the host, and antigen-antibody complexes. The differentiation pathways of the macrophage lineage are extremely complex, and macrophage phenotypes specific for different anatomical locations are found. The extent to which different macrophage phenotypes represent true developmental subpopulations, or alternatively simply represent the influence of the local microenvironment, remains largely unresolved. In addition, the phenotype of the macrophage is under complex regulatory control, via a host of soluble mediators produced by the specific and the innate immune system. The macrophage is not, primarily, an antigen-presenting cell. Thus expression of class I and class II MHC under resting conditions is low, and macrophages frequently suppress, rather than enhance, a T-cell-dependent immune response, via the release of inhibitory inflammatory mediators including arachidonic acid metabolites. The best-studied macrophage types are the peritoneal macrophages, particularly in rodents, and the circulating macrophage precursors, the blood monocytes in humans. In both these cell types the normal low levels of class I and class II MHC expression can be dramatically upregulated under the influence of T-cell-derived cytokines, particularly interferon-γ. T N F and GM-CSF also up-regulate MHC expression in these cell types. Under these conditions, activated macrophages can be demonstrated to show strong antigen-presenting function, a function which may be particularly important in processing and then presenting microorganisms relatively resistant to cellular degradation, such as many intracellular parasites. The regulatory relationship between T cells and macrophages is therefore complex and reciprocal - this is likely to be a key interaction underlying the immunopathology associated with chronic immunological responsiveness (13).

3.3. The B cell Antigen processing and presentation by B lymphocytes forms the molecular basis for T/B cooperation, and gives rise to the fundamental phenomenon of 'linked' help, whereby T-cell help for B-cell epitopes is restricted to those epitopes which physically form part of one molecular unit. Processing and presentation by B cells follows the same general rules as those of other cell types, with the important exception that antigen uptake and entry into the processing pathway

ANTIGEN PROCESSING AND PRESENTATION

185

is specifically promoted by interaction of antigen with antibody at the cell surface. Each B cell therefore takes up antigen specifically via its surface Ig at concentrations several orders of magnitude lower than uptake of nonspecific antigen. Once bound via surface Ig, antigen is internalized and processed normally, thus releasing internal antigen structures to interact with MHC class II molecules, and stimulate appropriate T-cell responses. This mechanism ensures that B cells can recognize antigen predominantly via interactions with conformational epitopes on the surface of protein antigens, but can then stimulate T cells specific for linear processed epitopes, which often lie within the globular protein structure. The uptake of antigen as a complex with surface Ig imposes additional constraints on the degradation and processing of antigen, since epitopes within or close to the antibody-binding sites may be partially protected from the action of proteinases (determinant protection) - thus the B-cell (antibody) repertoire may play a major role in shaping the repertoire of the T-cell response, and conversely, the nature of the dominant epitopes recognized by the T-cell pool will play a major role in shaping the B-cell repertoire by selecting which B cells will receive maximum 'help'. B-cell differentiation and antigen presentation B cells constitutively express low levels of MHC class II molecules, but resting B cells fail to express effective co-stimulator signals for T-cell activation and hence can induce nonresponsiveness or tolerance. In contrast, activation of B cells (via T-cell-derived cytokines, and accessory molecule interaction including the CD40/gp39 interaction) induces up-regulation of MHC class II, and simultaneous expression of strong co-stimulatory activity, including expression of the CD28 ligand B7. Thus activated B cells are potent antigen-presenting cells, at least for secondary immune responses. Further differentiation of B cells to plasma cells is accompanied by the shutting off of MHC class II synthesis (14).

4. INHIBITORS OF ANTIGEN PROCESSING A key to the dissection of the antigen-processing pathway has been the availability of specific inhibitors of individual steps of the pathway. The properties of the more commonly used inhibitors is shown in Table 8. A number of other inhibitors have been used occasionally, but the data on their action are too limited to include, and it is advisable to collaborate with a laboratory experienced in the analysis of such inhibitors, as new molecules are being developed constantly. It is important to appreciate that, although the inhibitors are usually used with the purpose of blocking one specific reaction in the antigen-processing pathway, this specificity is often not achieved. Thus chloroquine, which blocks acidification of intracellular organelles, not only inhibits protein degradation in these organelles but invariant chain degradation and thus MHC-peptide assembly. Similar caveats operate for all the molecules listed, and appropriate controls for such secondary effects must be included. There has obviously been very considerable interest in the possibilities of using inhibitors of processing/presentation as potential novel therapeutic approaches to autoimmunity or transplant rejection. So far, with the exception of chloroquine, which is partially effective in the treatment of rheumatoid arthritis, but whose mode of action in this disease is not known, no such strategy has proved feasible, perhaps because of the relatively nonspecific nature of many of the processes being targeted. In the absence of suitable small molecular weight inhibitors (still the molecules of choice for pharmaceutical development) much attention has focused on the use of biological response modifiers to block T-cell activation at the level of antigen presentation. One approach has used antibodies to cell-surface structures (e.g. CD4, CTLA-4, CD58), with some limited success in preliminary clinical trials. An alternative strategy has been to identify specific peptide analogs which can block the tripartite antigen-MHC-T-cell receptor interaction, either at the level of

186

CELLULAR IMMUNOLOGY LABFAX

Table 8. The pharmacological tools used in the study of antigen processing and presentation Molecule

MW (Da)

Source

Cycloheximide

281

Brefeldin A

Solubility

Working concn (μΜ)

Function

Ref.

Fungal Methanol, antibiotic ethanol

3-30

Inhibits protein synthesis, and hence especially de novo MHC class I synthesis

34,35

280

Fungal Methanol antibiotic

3-30

Inhibits egress from ER, and hence especially processing for class I M H C antigen presentation

36,37

Chloroquine

320

Synthetic organic heterocyclic

Water

100-500

Inhibits endosomal 38,39 acidification, and hence processing for class II M H C

Primaquine

259

Synthetic organic heterocyclic

Water, ethanol

100-500

As for chloroquine 40

Ammonium chloride

53

Inorganic

Water

10 000

As for chloroquine

Monensin

671

Fungal Ethanol antibiotic

30 000

As for chloroquine 42

Leupeptin

457

Fungal peptide

Water

10-100

Inhibits cysteine and some serine proteinases, and hence processing for class II M H C

Pepstatin

686

Fungal peptide

DMSO

10-100

43 Inhibits aspartic proteinases, and hence processing for class II M H C

E-64

357

Fungal peptide

DMSO

10-100

43 Inhibits cysteine proteinases and hence processing for class II M H C

41

43

Abbreviations: DMSO, dimethyl sulfoxide; E-64, iV-iV-L-3-transcarboxyoxizan-2-carbonyl-L-leucyl-agmatine.

ANTIGEN PROCESSING AND PRESENTATION

187

the binding of processed antigen to M H C , or by anergizing specific T-cell clones, or by blocking the subsequent interaction with the specific T-cell receptor. Some promising results have been obtained in vitro and in animal model systems, but the real potential of such strategies in terms of clinical value remains to be evaluated.

5. THE ANTIGEN-PROCESSING PATHWAY The dissection of the detailed cell biology of antigen processing has proved to be one of the most difficult aspects of the field. Significant progress in this field has occurred mainly within the past 5 years, and many details remain to be established. Nevertheless, the major elements of the intracellular pathways followed by antigen during processing are shown in Figure 2. The use of antigen-processing mutants, improved immunoelectron microscopy, pulse-chase metabolic labeling of proteins followed by immunoprecipitation, and transfection of genes coding individual components of the pathway into nonexpressing cell lines, have all proved particularly valuable techniques. Perhaps the most fundamental, and unexpected, finding has been the realization that class I and class II processing pathways are generally distinct (although division between the two pathways may not be quite as rigorous as was at first thought). In addition to this unexpected dichotomy, it is gradually becoming appreciated that antigen processing is a distinct cellular function, with its own components, and its own intracellular compartments, although making use of more general elements of cell physiology. Examples of such specializations are the TAP (transporter associated with antigen processing) genes of the class I pathway, and the 'MHC loading' compartment of the class II pathway. The outdated idea that antigen processing is simply an unregulated by-product of protein catabolism is slowly being abandoned. F i g u r e 2. T h e M H C processing pathways: (a) class I, (b) class I I .

(a)

Antigen degraded, possibly by ubiquitindependent proteosome action. Peptides transported across into ER via TAP transporter

Peptides associate with nascent MHC class I, and stabilize intact MHC/ß2 microglobulin complex

Complex expressed at the cell surface, where stable for many hours. Recognition by T cell may occur

188

Cytoplasm

Endoplasmic reticulum

Plasma membrane

CELLULAR IMMUNOLOGY LABFAX

6. THE MOLECULES The triumph of cellular immunology over the past decade has been its ability to dissect complex cellular phenomena into collections of well-characterized molecules. Nowhere has this progress been more apparent than in the fields of antigen processing and presentation. As a tribute to this effort, and perhaps to allow those who have become fixated on single molecules to adopt a more balanced approach, Table 9 lists all the molecules believed to play an important role in antigen processing or presentation. All the molecules listed have been cloned and sequenced, and in most cases also identified by specific antibody reactions. Analysis of the function of such molecules has remained much more difficult: approaches commonly used include trying to modulate antigen processing/presenting function by addition of blocking antibody to functional assays, or in the case of soluble molecules (cytokines) by addition of the molecule itself to the assays; by transfection of the relevant gene into appropriate negative cell lines; or, most recently, by the production of transgenic mice in which the relevant gene is either overexpressed, or expression is 'knocked-out' by homologous recombination. Once again, we have omitted those molecules whose involvement in antigen

Figure 2. (b)

Synthesis and export of class Il MHC and invariant chain, which form complexes in ER. Invariant chain prevents binding of peptides in ER, and directs transport of MHC away y from direct egress to the plasma membrane

Antigen taken up by receptor-mediated (especially Ig on B cells) or fluid-phase endocytosis. Limited proteolysis may occur on the membrane

Extracellular space

Some proteolysis of antigen may occur. Newly synthesized class II MHC/invariant chain trimers may passage through and be sorted in this compartment

Early endosome

Antigen further degraded. Invariant chain degraded, perhaps by cathepsin B. Processed antigen binds to MHC class II

Late endosome

Peptide/MHC complexes exported to plasma membrane. Some recirculation via early endosome may occur, but not likely to be functionally important

Plasma membrane

Cytoplasm and endoplasmic reticulum

►

ANTIGEN PROCESSING AND PRESENTATION

P.

193

189

Table 9. The molecular components of the antigen-processing and presentation pathways Molecule

Other names

Intracellular Cathepsin B Cathepsin D Cathepsin E

Invariant chain

M W (kDa)

Main function in antigen presentation

Ref.

27

Cysteine proteinase involved in antigen and/or invariant chain degradation Aspartic lysosomal proteinase involved in antigen degradation Aspartic nonlysosomal proteinase, esp. in gastrointestinal epithelium; role in antigen processing Binds to, and regulates the folding and transport of M H C class II molecules; also prevents binding of processed peptides to class II M H C in the endoplasmic reticulum Proteosome subunit, perhaps involved in antigen degradation for class I M H C processing As for LMP1 Peptide transporter subunit, carrying processed peptides from cytoplasm into the ER, for presentation by class I M H C As for TAPI

43

gp 38-40 Slowmoving aspartic protease CD74

gp 42/84

gp 43/41/35/33 iLsoforms

LMP1 LMP2 TAPI

TAP2 ell-surface molecules M H C class I M H C class II DMA DMB Ig B7

190

a gp 44 ß2m p l 2 dimer a gp 35 ßgp28 dimer

gp 150-185 BB1

60 gp

Peptide-binding molecules presenting antigen to CD8 T cells Peptide-binding molecules presenting antigen to CD4 T cells Nonpolymorphic class II homolog. Function unknown As for DMA Antigen-specific receptor mediating uptake and processing of antigen by B lymphocytes Expressed on activated B cells, and professional antigenpresenting cells, and providing co-stimulatory activity to T cells via interaction with its ligands CD28 or CTLA-4 on T cells

44 45

46

47 47 48

48 49 10 50 50 14 51

CELLULAR IMMUNOLOGY LABFAX

Table 9. Continued Molecule CTLA-4 HSP-70 CD1 CD2

CD3-TCR complex

CD4 CD5 CD8 CD 11a

CD13 CD 14 CD18 CD23

CD28 CD40

Other names

MW (kDa)

Main function in antigen presentation

Ref.

2 x gp 26 homodimer p70

SeeB7

21

Peptide-binding protein facilitating antigen-MHC interaction Putative peptide-presenting gp 43-49 molecule for γ/δ Τ cells gp50 Adhesion molecule on T cells, binding CD58 (LFA-3) on antigen-presenting cells, and enhancing antigen presentation CD3: γ gp 26, T-cell antigen-specific receptor δ gp 20, ε p 20, complex, interacting with MHC-peptide complex and ζ ρ16, η p 22 TCR: a gp 40-45, providing specificity element to T cell-antigen-presenting cell ß gp 38-45 interaction gp55 M H C class II binding receptor on T cells, enhancing antigen presentation Marker of a subset of B cells, that gp67 is particulary effective in antigenpresenting function M H C class I binding receptor on agp32 T cells, enhancing antigen ßgp32 presentation dimer One chain of the LFA-1 complex, LFA-1, gp 180 which binds ICAMs, and forms a chain an adhesive interaction between to CD 18 T cell and antigen-presenting cell, which enhances antigen presentation Cell-surface aminopeptidase, which Aminogpl50 may have a role in trimming peptidase MHC-bound pep tides N Macrophage marker; receptor for the gp55 LPS-LPS binding protein complex See C D l l a β chain to g p 9 5 CDlla (LFA-1) IgE Fc receptor; putative role in gp45-5 FCERII modulating antigen processing and presentation of antigens for IgE production See B7 2 x gp 44 homodimer Receptor for gp39 on T cells, gp50 regulating B-cell activation and survival

52

ANTIGEN PROCESSING AND PRESENTATION

53 51

15 51 15 51

54 51 55

17 56

191

Table 9. Continued Molecule

Other names

MW (kDa)

Main function in antigen presentation

Ref.

CD44

Pgp-1

gp 85-250

Down-regulates the adhesive interaction between T cells and dendritic cells Binds to an unspecified ligand, and down-regulates antigen presentation via its cytoplasmic tyrosine phosphatase activity See CD 11a

57

CD45 (RA,B andO) CD54 CD55 CD58 CD59 CD64 Cytokines IFN-a and -ß

gp 180-221

ICAM-1 DAF LFA-3 FCyRI

gp 85-110 gp 64-73 gp 56-70 gp 18-20 gp75

gp 17-40

IFN-γ

2 x gp 20-25 homodimer

TNF-α and ß

a: 3 x p 17 homotrimer ß: 3 x gp 25 homotrimer

I L - l a and ß

gpl7

IL-4

gp20

IL-6

gp26

IL-10

2 x p 18 homodimer

GM-CSF

gp 14-35

See CD2 Modulates antigen processing via its effect on antigen uptake Up-regulate class I M H C (occasionally also class II) Converts macrophages to efficient antigen-presenting cells by up-regulating expression of M H C and a variety of adhesion molecules; also induces M H C on a variety of other cell types Synergizes with IFN-γ, in regulating macrophage function; regulates M H C expression; regulates Langerhans cell/dendritic cell differentiation Co-stimulatory molecules produced by macrophages, and enhancing antigen presentation; regulates differentiation along the Langerhans cell/dendritic cell pathway Up-regulates class II M H C on B cells and macrophages Macrophage derived co-stimulator (with IL-1) of T-cell activation Inhibits the ability of antigenpresenting cells to stimulate a TH-1 type cytokine response Regulates differentiation along the Langerhans cell/dendritic cell pathway, and also along the monocyte/macrophage pathway; in both cases enhances class I M H C expression

57

51 57 51 58 4

59 59

60, 61

62

63 64 65 66

LMP, low molecular mass polypeptide complex.

192

CELLULAR IMMUNOLOGY LABFAX

processing is either very indirect, or inadequately documented at the time of writing. Data on the function of many of the molecules continue to be collected apace, and inaccuracies are bound to be revealed. Finally, most of the molecules of the immune system are pleiotropic, and the functions briefly listed in Table 9 are meant as a guide to their involvement in antigen presentation only.

7. CONCLUSIONS The past 10 years have seen an explosive growth in the study of antigen processing and presentation. In common with many other branches of biology, the impact of molecular biology has had a disproportionate effect, leading to the identification of several dozen molecules directly involved. For this reason, the focus of this collection has been molecular molecular genetics, molecular analysis of antigens and molecular dissection of antigenpresenting systems. There is clearly considerably more of this type of analysis to come, and the number of molecules likely to be uncovered may well double or triple in the next 10 years. A different type of analysis of antigen presentation/processing has hardly begun, however; by this is meant the synthesis of the disparate components of the system into an integrated model of the whole, and more particularly, the ability to identify rules which govern the behavior of the system in respect to its parts. An example of the type of question to be addressed would be the impact of antigen-processing enzyme specificity on the repertoire of the T-cell response, or at least on the repertoire of the set of MHC-bound pep tides. Another would be an analysis of the interactive hierarchy of co-stimulator signals which govern the antigen-processing event - what is the contribution of each, relative to the contribution to each other, and what governs the relative change in contribution of each, in relation to changes in activity of each other? Many other such integrative questions remain to be answered.

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